GROWTH RATE, PATHOGENICITY AND FUNGICIDE SENSITIVITY OF Macrophomina spp. FROM WEEDS, MELON AND WATERMELON ROOTS

ESTUDO DO CRESCIMENTO MICELIAL DE MACROPHOMINA SPP. NO BRASIL, SUA PATOGENICIDADE E SENSIBILIDADE A FUNGICIDA

ABSTRACT

Macrophomina (Botryosphaeriaceae) is one of the main genera of soilborne phytopathogenic fungi, which causes root and seed rot in more than 800 host plants worldwide. Recent phylogenetic studies identified the species M. phaseolina and M. pseudophaseolina in Trianthema portulacastrum and Boerhavia diffusa in melon and watermelon production areas in northeastern Brazil. Therefore, the objective of this study was: i) to verify the effect of temperature and salinity on the mycelial growth of M. phaseolina, M. pseudophaseolina and M. euphorbiicola, ii) to assess their pathogenicity on melon and watermelon seedlings, and iii) to determine their sensitivity to the fungicide carbendazim. The optimal temperature for mycelial growth rate (MGR) for Macrophomina spp. ranged from 27.18 ºC (CMM4771 – M. pseudophaseolina) to 31.80 ºC (CMM4763 – M. phaseolina). For the effect of salinity on mycelial growth of Macrophomina isolates, the EC₅₀ ranged from 103.76 (CMM4868 – M. euphorbiicola) to 315.25 mM (CMM4801 – M. pseudophaseolina). The pathogenicity test demonstrated that M. phaseolina, M. pseudophaseolina and M. euphorbiicola are pathogenic on melon with M. phaseolina exhibiting a higher level of virulence. Macrophomina euphorbiicola isolates did not cause disease in watermelon. The most sensitive isolates to the fungicide carbendazim were CMM4868, CMM4867 (M. euphorbiicola) and CMM1531 (M. phaseolina) with EC₅₀ of 0.003, 0.012 and 0.012 mg.L^-1 a.i., respectively. All Macrophomina spp. used in these experiments were pathogenic to the tested melon and watermelon cultivars with the exception of the M. euphorbiicola isolate that did not cause damage to watermelon.

Keywords:
Citrullus lanatus; Cucumis melo; Salinity; Soilborne fungi; Temperature

RESUMO

Macrophomina (Botryosphaeriaceae) é um dos principais gêneros de fungos fitopatogênicos de solo, que causam apodrecimento de raízes e sementes em mais de 800 plantas hospedeiras em todo o mundo. Estudos filogenéticos recentes identificaram as espécies M. phaseolina e M. pseudophaseolina em Trianthema portulacastrum e Boerhavia diffusa em áreas de produção de melão e melancia no Nordeste do Brasil. Portanto, o objetivo deste estudo foi: i) verificar o efeito da temperatura e salinidade sobre o crescimento micelial de M. phaseolina, M. pseudophaseolina e M. euphorbiicola, ii) avaliar sua patogenicidade em mudas de melão e melancia, e iii) determinar suas sensibilidades ao fungicida carbendazim. A temperatura ótima para taxa de crescimento micelial (MGR) para Macrophomina spp. variou de 27,1 ºC (CMM4771 – M. pseudophaseolina) a 31,8 ºC (CMM4763 – M. phaseolina). Para o efeito da salinidade no crescimento micelial de isolados de Macrophomina, a EC₅₀ variou de 103,76 (CMM4868 – M. euphorbiicola) a 315,25 mM (CMM4801 – M. pseudophaseolina). O teste de patogenicidade demonstrou que M. phaseolina, M. pseudophaseolina e M. euphorbiicola são patogênicas em melão com M. phaseolina apresentando maior virulência. Isolados de Macrophomina euphorbiicola não causaram doenças em melancia. Os isolados mais sensíveis ao fungicida carbendazim foram CMM4868, CMM4867 (M. euphorbiicola) e CMM1531 (M. phaseolina) com EC₅₀ de 0,003; 0,012 e 0,012 mg.L^-1 i.a., respectivamente. Todas as espécies de Macrophomina spp. utilizados nestes experimentos foram patogênicos para as cultivares de melão e melancia testadas com exceção dos isolados de M. euphorbiicola que não causaram danos à melancia.

Palavras-chave:
Citrullus lanatus; Cucumis melo; Salinidade; Fungos habitantes do solo; Temperatura

INTRODUCTION

Macrophomina phaseolina (Tassi) Goid. (GOIDÀNICH, 1947) (Botryosphaeriaceae, Ascomycota) is a soil-borne pathogenic fungus with a worldwide distribution on over 800 species of plant including economically important hosts such as soybean [Glycine max (L) Merrill], melon (Cucumis melo L.), watermelon [Citrullus lanatus (Thunb.) Matsum. & Nakai], common bean (Phaseolus vulgaris L.), cowpea (Vigna unguiculata L. Walp), sorghum (Sorghum bicolor L.) and cotton (Gossypium herbaceum L.) (SALES JÚNIOR et al., 2020; LODHA; MAWAR, 2020; FARR; ROSSMAN, 2022). It causes different diseases: charcoal rot, grey stem rot, root rot, damping-off and seed damage, being more aggressive in tropical and subtropical countries, with a semiarid climate. In addition, the ability of this pathogen to survive in soil and/or crop residues through resistance/survival structures (microsclerotia), as well as in seeds, makes its management quite difficult (DHINGRA; SINCLAIR, 1978).

For a long time, it was considered that only one Macrophomina species (M. phaseolina) existed, however, phylogenetic studies, carried out in the last decade, have shown the existence of high genetic variability in the genus Macrophomina (SARR et al., 2014; MACHADO et al., 2019; ZHAO et al., 2019). Consequently, four new species of Macrophomina have been recently described: M. pseudophaseolina Crous, Sarr and Ndiaye found in crops of Abelmoschus esculentus (L.) Moench., Arachis hypogaea L., Hibiscus sabdariffa L. and Vigna unguiculata (L.) Walp. in Senegal (SARR et al., 2014), A. hypogaea, Gossypium hirsutum L., Ricinus communis L., Jatropha curcas L. (MACHADO et al., 2019) and Manihot esculenta C. (BRITO et al., 2019), and in weeds of Boerhavia diffusa L. and Trianthema portulacastrum L. in Brazil (NEGREIROS et al., 2019); M. euphorbiicola A.R. Machado, D.J. Soares and O.L. Pereira in R. communis and Jatropha gossypifolia L. in Brazil (MACHADO et al., 2019); M. vaccinii Y. Zhang ter and L. Zhao in Vaccinium spp. in China (ZHAO et al., 2019); and more recently, M. tecta Vaghefi, B. Poudel & R.G. Shivas in S. bicolor in Australia (POUDEL et al., 2021). The study of adaptability components, widely used for new species of fungi, such as sensitivity to salinity and fungicide, mycelial growth at different temperatures and virulence has been very useful for evaluating the variability of the adaptability of isolates in populations of plant pathogenic fungi. However, as adaptability has a relative character, it must be estimated by measuring characters that reduce some adaptive advantage among individuals (CORREIA et al., 2014). In addition, the competitive capacity among populations of fungi can be inferred indirectly through these adaptability components (ZHAN; McDONALD, 2013). Thus, data on pathogenicity and adaptability of isolates can directly influence the management measures to be adopted in field production (MENGISTU et al., 2018).

So far, studies on adaptability components comparing the different Macrophomina spp. currently occurring in Brazil are scarce. Therefore, this work aims to investigate the characterization and pathogenicity of M. phaseolina, M. pseudophaseolina and M. euphorbiicola isolates obtained from T. portulacastrum and B. diffusa collected in northeast Brazil, regarding: i) the effect of temperature and salinity on mycelial growth, ii) their pathogenicity on melon and watermelon seedlings, and iii) sensitivity to the carbendazim fungicide.

MATERIAL AND METHODS

Fungal isolates

In this study, eight Macrophomina isolates were used. Six isolates of three species of Macrophomina (M. phaseolina – CMM4738 and CMM4763, M. pseudophaseolina – CMM4771 and CMM4801; and M. euphorbiicola – CMM4867 and CMM4868) from asymptomatic roots of the weed species T. portulacastrum and B. diffusa, collected in melon and watermelon fields located in the Rio Grande do Norte (RN) and Ceará (CE) states, northeastern Brazil; and two isolates of M. phaseolina, collected from a melon (CMM1531) and watermelon (MC01) roots, were used as positive controls in the experiments (Table 1).

Table 1
List of Macrophomina species used in this study.

Macrophomina species	Isolate numbera	Hostc	Locationb	GenBank Accession Numbers
M. phaseolina	CMM4738	TP	Brazil, CE, Icapuí	MH373461
	CMM4763	BD	Brazil, RN, Mossoró	MH373451
M. pseudophaseolina	CMM4771	TP	Brazil, RN, Assú	MH373471
	CMM4801	BD	Brazil, RN, Assú	MH373517
M. euphorbiicola	CMM4867	TP	Brazil, RN, Assú	MH712509
	CMM4868	BD	Brazil, RN, Assú	MH712510
M. phaseolina (Positive Control - PC)	CMM1531	CM	Brazil, RN, Mossoró	MN136199
	MC01	CL	Brazil, CE, Icapuí	MN136200

• ^aCulture Collection of Phytopathogenic Fungi “Prof. Maria Menezes” (CMM) of the Universidade Federal Rural de Pernambuco (Recife, PE, Brazil). ^bCeará state (CE) and Rio Grande do Norte state (RN). ^c TP - Trianthema portulacastrum. BD - Boerhavia diffusa. CM - Cucumis melo. CL - Citrullus lanatus.

The CMM4867 and CMM4868 isolates of M. euphorbiicola and CMM1531 and MC01 of M. phaseolina were identified through phylogenetic inference based on the partial sequence of the translation elongation factor-1alpha (tef-1α) using the primers EF728F and EF986R (CARBONE; KOHN, 1999). The others isolates of Macrophomina spp. were identified by Negreiros et al. (2019).

The isolates (CMM4738, CMM4763, CMM4771, CMM4801, CMM4867, CMM4868 and CMM1531) were deposited in the Culture Collection of Phytopathogenic Fungi “Prof. Maria Menezes” (CMM) at the Universidade Federal Rural de Pernambuco (Recife, Pernambuco, Brazil). The isolate (MC01) was deposited in the culture collection of plant pathogenic fungi of Universidade Federal Rural do Semi-Árido (Mossoró, Rio Grande do Norte, Brazil). All isolates were hyphal‐tipped and, then, they were stored on sandy‐organic substrate and Castellani’s method with distilled water (NEGREIROS et al., 2019).

Effects of temperature on mycelial growth of Macrophomina

The mycelial growth gate (MGR) was measured in colonies grown in Petri plates containing potato-dextrose-agar (PDA) (MAYEK-PÉREZ; LÓPEZ-CASTAÑEDA; ACOSTA-GALLEGOS, 1997). Mycelial plugs (8 mm in diameter) obtained from the growing edge of 7-day-old colonies of isolates were placed in the centre of each Petri plate (one plug per plate), which were then incubated at the temperatures of 25, 30, 35, 40 and 45 ± 1 °C, in the dark, for seven days. The colony diameter of each isolate for all temperatures was measured daily along two perpendicular axes until the colony reached the edge of the Petri plate and the data were used to calculate the MGR of the colony (cm day^-1).

Effect of salinity on mycelial growth of Macrophomina spp.

The effect of salinity on mycelial growth of all isolates in vitro was analysed using PDA adjusted to the following sodium chloride (NaCl) concentrations: 0, 250, 500, 750 and 1000 mM (corresponding to 0.82, 3.70, 5.57, 7.59 and 9.36 dS m^–1, respectively). All concentrations were sterilized prior to its use in autoclave for 15 min, at 121 ºC and 1 Bar. Mycelial discs (8 mm in diameter) taken from 7-day old fungal colonies of each isolate were transferred to Petri plates containing PDA adjusted to each NaCl concentration, and incubated at 30 ºC in the dark. The average diameter of the fungal colony was measured daily and data were used to calculate the MGR of the colony.

Pathogenicity of Macrophomina spp. on melon and watermelon seedlings

The pathogenicity of Macrophomina spp. was evaluated on melon “Gladial” and watermelon “Crimson sweet” seedlings. Melon and watermelon seeds were germinated in pots containing Tropstrato HT® commercial substrate previously autoclaved. The seedlings were daily irrigated to drainage with tap water. For inoculation the toothpick method was used, because of the easy multiplication of inoculum and fast inoculation (NEGREIROS et al., 2019). Melon and watermelon seedlings were inoculated 10 days after sowing (DAS) by inserting the toothpicks colonized with mycelia and microsclerotia of the corresponding isolate in each hypocotyl, 1 cm above the soil. Non-infested and autoclaved toothpicks were used as negative controls.

The inoculated seedlings were maintained in a greenhouse at an average temperature of 35 ± 2 °C, under natural daylight conditions using a completely randomized experimental design, with five replicates per treatment (isolate). Thirty days after inoculation, disease incidence was determined as the total number of infected plants from each Macrophomina species and expressed as a percentage. The aggressiveness of the isolates was assessed as disease severity using a modified version of the rating scale described by Ambrósio et al. (2015), where, 0 = symptomless, 1 = less than 3% of shoot tis sues infected, 2 = 3–10% of shoot tissues infected, 3 = 11–25% of shoot tissues infected, 4 = 26–50% of shoot tissues infected and 5 = more than 50% of shoot tissues infected.

Sensitivity of Macrophomina spp. to carbendazim

The sensitivity of the three Macrophomina species to the fungicide carbendazim (methyl-2-benzimidazole carbamate, benzimidazoles chemical group) was determined in PDA supplemented with carbendazim, and the MGR was evaluated. The treatments included five levels of carbendazim concentration: 0.01, 0.10, 1, 10 and 100 mg L^-1 active ingredient (a.i.) (TONIN et al., 2013). Petri plates containing PDA without fungicide were used as the control (0 mg L^-1). A 7-day old mycelial plug (8 mm in diameter) from each isolate of Macrophomina was placed in the centre of the Petri plates containing PDA supplemented with the concentrations of the fungicide, and incubated in the dark at 30 °C. The radial growth (diameter) of each colony was measured daily in two perpendicular directions, until the colony reached the edge of the Petri plate and the mean diameter of the colony was obtained.

Relationships between adaptability components and pathogenicity of Macrophomina spp.

To establish the relationship between adaptability components and pathogenicity of Macrophomina spp., a heatmap and principal component analysis (PCA) were performed. The standardized Euclidean distance between species pairs was used to construct a heatmap (dendrogram) by unweighted paired group method with arithmetic averages (UPGMA) (KOLDE, 2022) and the PCA (LE; JOSSE; HUSSON, 2008) was performed by correlation matrix, both using the software “R” (R CORE TEAM, 2022).

Statistical analysis

All experiments were conducted in a completely randomized design, with five repetitions of each isolate. All experiments were repeated. Preliminary ANOVAs were performed to determine if there were significant differences between the repetitions of the experiments and whether the data could be combined. The MGR means of temperature and NaCl concentrations of all isolates were subjected to a regression analysis using TableCurve 2D v.5.01 (SYSTAT Software Inc.). For each temperature and NaCl concentration, the means of the isolate were compared by Tukey test at the 5% significance level using the software “R” (R CORE TEAM, 2022). Differences in incidence and severity caused by Macrophomina species for melon and watermelon were analysed with the non-parametric Kruskal-Wallis test at the probability level of 5% (p < 0.05) using the software “R” (R CORE TEAM, 2022). The MGR of the fungicide was used to determine the effective concentration for 50% reduction in growth – EC₅₀ (mg L^-1 of a.i. inhibiting MGR by 50%) for each isolate of Macrophomina and the method was based on linear regression by plotting values of Log-Probit.

For all experiments, no significant effect of the experiment repetitions (ANOVA, p > 0.05) was found, thus the data were combined.

RESULTS AND DISCUSSION

Effects of temperature on mycelial growth of Macrophomina spp.

In the temperature study, Macrophomina spp. showed statistically significant effects (p ≤ 0.05) for fungal MGR. The optimum temperature for the three Macrophomina spp. ranged from 27.18 ºC (CMM4771 – M. pseudophaseolina) to 31.80 ºC (CMM4763 – M. phaseolina), being the mean MGR of the isolates 29.57 ºC (Table 2). The M. phaseolina isolates (CMM4738, CMM4763, CMM1531 and MC01) showed the optimum temperature of 28.51, 31.80, 29.10 and 28.09 ºC, respectively. However, the optimum temperature for M. pseudophaseolina isolates (CMM4771 and CMM4801) was 27.18 and 31.32 ºC, respectively, and for M. euphorbiicola isolates (CMM4867 and CMM4868) was 28.89 and 31.65 ºC, respectively. The MGR of the M. euphorbiicola isolates (CMM4867 and CMM4868) and M. phaseolina (CMM1531) at 25 ºC and 30 ºC was significantly higher than those of other isolates. At 35 ºC and 40 ºC, the MGR of the isolate CMM4868 (M. euphorbiicola) was significantly higher than other isolates. No growth was observed at 45 °C for any of the three Macrophomina species isolates evaluated in this study after seven days of incubation.

Table 2
Optimum temperature and mycelial growth at 25, 30, 35 and 40 ºC of Macrophomina spp. from northeastern Brazil.

Macrophomina species	Isolates	Temperature
		Optimum temperature (°C)	Mycelial Growth Rate (cm d-1)b
			25ºC	30ºC	35ºC	40ºC
M. phaseolina	CMM4738	28.51	1.07 bc	1.39 b	0.70 d	0.35 cde
	CMM4763	31.80	0.63 e	1.34 b	1.59 b	0.67 b
M. pseudophaseolina	CMM4771	27.18	0.83 cde	0.88 d	0.44 e	0.22 e
	CMM4801	31.32	0.74 de	0.86 d	0.97 c	0.57 b
M. euphorbiicola	CMM4867	28.89	1.37 a	1.93 a	0.97 c	0.48 bcd
	CMM4868	31.65	1.20 ab	2.05 a	1.94 a	1.34 a
M. phaseolina (PCa)	CMM1531	29.10	1.37 a	2.05 a	1.03 c	0.51 bc
M. phaseolina (PCa)	MC01	28.09	0.91 cd	1.09 c	0.54 de	0.27 de
Mean		29.57	1.01	1.45	1.02	0.55
CV (%)		-	11.71	5.79	9.28	16.41

• ^a PC (Positive control). ^b Values with the same letter within a column are not significantly different according to the Tukey test at 5% probability.

In this study, optimal growth temperatures for the isolates of Macrophomina spp. ranged between 27.18 and 31.80 °C, which differs from previously published results by Sarr et al. (2014). These authors studying the genetic diversity of M. phaseolina in Senegal, reported for M. phaseolina and M. pseudophaseolina optimal growth temperature in the range of 30 to 36 ºC and there was still growth at 40 ºC. In a previous study, Cardona (2006) reported the ideal temperature for M. phaseolina in Venezuela as in the range of 28 to 32 ºC, our results are within this range. Previously, Csöndes (2012) reported that the most favourable temperature interval for the development of M. phaseolina isolates in Hungary was from 25 to 35°C. The number of studies concerning optimal temperatures for maximal growth of M. pseudophaseolina and M. euphorbiicola is even more limited in relation to the importance that it can have for effective management of the disease. The occurrence of Macrophomina spp. in growing areas of melon and watermelon in the northeastern semi-arid region might be related to the hot and dry climate of the region (INMET, 2022), with average annual temperatures ranging from 23.2–33.8 ºC, consequently, Macrophomina is one of the most frequent root pathogens isolated from symptomatic melon and watermelon plants.

Effect of salinity on mycelial growth of Macrophomina spp.

The mean NaCl concentrations of the Macrophomina spp. isolates were subjected to a regression analysis with a correlation coefficient R² > 0.99, and showed statistically significant effects (p < 0.05). The EC₅₀ for all Macrophomina isolates ranged from 103.76 (CMM4868 – M. euphorbiicola) to 315.25 mM (CMM4801 – M. pseudophaseolina), with a mean EC₅₀ of the isolates of 192.97 mM (Table 3). The M. phaseolina isolates showed EC₅₀ of 175.12 (CMM4738), 132.09 (CMM4763), 219.81 (CMM1531) and 193.06 mM (MC01). However, the EC₅₀ for M. pseudophaseolina isolates (CMM4771 and CMM4801) was 194.08 and 315.25 mM, respectively, and for M. euphorbiicola isolates (CMM4867 and CMM4868) was 210.56 and 103.76 mM, respectively. Statistically significant effects of the isolates of Macrophomina spp. for each NaCl concentration on MGR were observed (p < 0.05). At 0 mM (control), the mean MGR of the isolates was 1.22 cm d^-1 and the values of this variable ranged from 0.83 (CMM4771) to 2.05 cm d^-1 (CMM4868). At 250 mM, the mean MGR was 0.35 cm d^-1 and the values ranged between 0.21 (MC01) and 0.64 cm d^-1 (CMM4867). This concentration showed a 71.31% reduction in the mean MGR in relation to the concentration of 0 mM (control). At 500 mM, the mean MGR was 0.15 cm d^-1, the values ranged between 0.08 (CMM4771) and 0.24 cm d^-1 (CMM1531), and 87.70% of reduction of the mean MGR in relation to the concentration of 0 mM. At 750 mM, the mean MGR was 0.09 cm d^-1, the values ranged between 0.02 (CMM4868) and 0.21 cm d^-1 (CMM1531), and this concentration showed 92.62% of reduction in the MGR in relation to the concentration of 0 mM. At 1000 mM, the mean MGR was 0.05 cm d^-1, the values ranged between 0.01 (CMM4867) and 0.09 cm d^-1 (CMM1531), and showed 95.90% of reduction of the MGR in relation to the concentration of 0 mM.

Table 3
Effective concentration for 50% reduction in growth (EC₅₀) and mean mycelial growth at 0, 250, 500, 750, and 1000 mM of sodium chloride of Macrophomina spp. from northeastern Brazil.

Macrophomina species	Isolates	Salinity
		Regression equationb	EC50 (mM)	Mycelial Growth Rate (cm d-1)c
				0	250	500	750	1000
M. phaseolina	CMM4738	y = 0.0668-0.0039x	175.12	1.07 c	0.35 bcd	0.16 b	0.08 bcd	0.05 b
	CMM4763	y = 0.2849-0.0052x	132.09	1.34 b	0.23 cd	0.11 cd	0.07 bcde	0.03 bcd
M. pseudophaseolina	CMM4771	y=-0.2118-0.0034x	194.08	0.83 d	0.27 cd	0.08 d	0.11 b	0.05 b
	CMM4801	y=-0.0151-0.0034x	315.25	0.86 d	0.38 bc	0.15 bc	0.05 cde	0.04 bc
M. euphorbiicola	CMM4867	y=0.3214-0.0034x	210.56	1.37 b	0.64 a	0.18 b	0.05 de	0.01 d
	CMM4868	y=0.7127-0.0066x	103.76	2.05 a	0.26 cd	0.14 bc	0.02 e	0.02cd
M. phaseolina (PCa)	CMM1531	y=0.2928-0.0031x	219.81	1.37 b	0.50 ab	0.24 a	0.21 a	0.09 a
M. phaseolina (PCa)	MC01	y=-0.1164-0.0034x	193.06	0.91 cd	0.21 d	0.14 bc	0.10 bc	0.08 a
Mean			192.97	1.22	0.35	0.15	0.09	0.05
CV (%)		-	-	6.64	19.49	12.16	23.84	21.92

• ^a PC (Positive control).
• ^by = mycelial growth rate; x = salinity concentration.
• ^cValues with the same letter within a column are not significantly different according to the Tukey test at 5% probability.

Sodium chloride reduced in vitro growth of Macrophomina species, particularly in isolate CMM4868 (M. euphorbiicola); while the isolate CMM4801 (M. pseudophaseolina) was the most tolerant to sodium chloride. Sodium chloride caused the greatest negative effects on the development of Macrophomina spp. Variations in response to salinity in the mycelial growth of fungi are not unusual, but for the species M. pseudophaseolina and M. euphorbiicola these data are scarce, therefore, the data from this study show this variation between isolates and within the Macrophomina genus. Similar results have been reported in M. phaseolina by Cervantes-García et al. (2003) and Fusarium solani (Mart.) Sacc. (PALACIOS et al., 2014). Negative effect by NaCl on Macrophomina spp. growth may be related to the modification of water availability in the PDA medium; therefore, the osmotic potential is lower in each fungal cell compared with the conditions of the PDA. The NaCl present in the PDA medium traps water molecules, therefore water will not be available to the isolates of Macrophomina. The energy spent by the fungus in order to obtain water molecules from the medium is increased as the NaCl concentrations in the PDA increase. Thus, the fungus is obliged to reduce its growth rate under in vitro conditions (CERVANTES-GARCÍA et al., 2003).

Pathogenicity of Macrophomina spp. on melon and watermelon

All isolates of Macrophomina were pathogenic to melon seedlings, but for the watermelon seedlings only the isolates CMM4801 (M. pseudophaseolina), CMM4763 and MC01 (M. phaseolina) were pathogenic (Table 4). The results showed that the incidence and severity of disease presented significant differences (p ≤ 0.05) for the isolates of M. phaseolina, M. pseudophaseolina and M. euphorbiicola, in each culture. In melon seedlings, the isolates CMM4738, CMM4763 and CMM1531 (M. phaseolina) were statistically different to the isolate CMM4868 (M. euphorbiicola) for disease incidence, presenting the highest averages 100%, 100% and 100%, respectively. However, for disease severity, only isolates CMM4771 (M. pseudophaseolina) and CMM4868 (M. euphorbiicola) differed from CMM1531 (M. phaseolina), the other isolates did not differ from each other. The isolates of M. pseudophaseolina and M. euphorbiicola showed intermediate values ranging from 0.8 (CMM4771 and CMM4868) to 3.8 (CMM4867) for severity, and from 20% (CMM4868) to 80% (CMM4801 and CMM4867) for disease incidence to melon. In watermelon seedlings, the isolates CMM4763 (M. phaseolina) and CMM4801 (M. pseudophaseolina) were not statistically different from the MC01 (M. phaseolina – PC) for disease incidence and severity, the isolates showed the same incidence and severity values of 40% and 0.4, respectively, to watermelon.

Table 4
Disease severity and incidence induced to Cucumis melo and Citrullus lanatus seedlings by Macrophomina spp. from northeastern Brazil.

Macrophomina species	Isolates	Cucumis melo				Citrullus lanatus
		Disease incidence (%)		Disease severity		Disease incidence (%)		Disease severity
		Rankb	Mean	Rankb	Mean	Rankb	Mean	Rankb	Mean
M. phaseolina	CMM4738	22.5 b	100	25.6 ab	4.8	13.5 a	0	13.5 a	0.0
	CMM4763	22.5 b	100	25.1 ab	4.6	20.5 ab	40	19.9 ab	0.4
M. pseudophaseolin a	CMM4771	12.0 ab	40	8.1 a	0.8	13.5 a	0	13.5 a	0.0
	CMM4801	19.0 ab	80	10.8 ab	1.4	20.5 ab	40	19.9 ab	0.4
M. euphorbiicola	CMM4867	19.0 ab	80	21.0 ab	3.8	13.5 a	0	13.5 a	0.0
	CMM4868	8.5 a	20	7.9 a	0.8	13.5 a	0	13.5 a	0.0
M. phaseolina (PCa)	CMM1531	22.5 b	100	27.5 b	5.0	-	-	-	-
M. phaseolina (PCa)	MC01	-	-	-	-	31.0 b	100	32.2 b	3.4
	Mean	-	74.3	-	3.0	-	26.0	-	0.6
	χ2 d	15.7	-	24.5	-	21.8	-	23.6	-

• ^aPositive control (PC).
• ^bχ² = chi-square value significant at 5% by Kruskal-Wallis test.

The pathogenicity test demonstrated that M. phaseolina, M. pseudophaseolina and M. euphorbiicola are pathogenic to melon with M. phaseolina exhibiting a higher level of aggressiveness. However, for watermelon, only CMM4801 (M. pseudophaseolina), CMM4763 and MC01 (M. phaseolina) isolates were able to cause disease with MC01 isolate exhibiting a higher level of virulence in the experiment. In this study, M. euphorbiicola isolates did not cause disease in watermelon. In a previous study, Ndiaye et al. (2015) investigating the pathogenicity of M. phaseolina and M. pseudophaseolina on three varieties of cowpea, observed that both species of Macrophomina induced disease. Negreiros et al. (2019) studying the pathogenicity of M. phaseolina and M. pseudophaseolina from weed species on melon seedlings revealed that all M. phaseolina isolates inoculated were able to cause disease to melon seedlings, but only some M. pseudophaseolina isolates were able to infect them.

Sensitivity of Macrophomina spp. to carbendazim

The effects of different concentrations of carbendazim on MGR of Macrophomina are shown in Table 5. Regression equations for log-Probit were adjusted and the EC₅₀ values were calculated. The mean EC₅₀ was 0.034 mg L^-1 a.i. and the values of this variable ranged from 0.003 (M. euphorbiicola) to 0.089 (M. pseudophaseolina) mg L^-1 a.i. of carbendazim (Table 5). The most sensitive isolates to the fungicide carbendazim were CMM4868, CMM4867 (M. euphorbiicola) and CMM1531 (M. phaseolina) with EC₅₀ of 0.003, 0.012 and 0.012 mg L^-1 a.i., respectively. In contrast, the most tolerant species was M. pseudophaseolina (CMM4771 and CMM4801) with EC₅₀ of 0.060 and 0.089 mg L^-1 a.i., respectively.

Table 5
Regression equation and effective concentration for 50% reduction in growth (EC₅₀) for log-Probit analysis by fungicide carbendazim by Macrophomina spp. from northeastern Brazil.

Macrophomina species	Isolates	Fungicide Carbendazim
		Regression equationb	EC50c (mg L-1 a.i.)
M. phaseolina	CMM4738	y=18.2693252040x+75.2023275931	0.042
	CMM4763	y=15.4255583900x+77.5926546017	0.016
M. pseudophaseolina	CMM4771	y=18.7431597070x+72.8887478025	0.060
	CMM4801	y=19.6776603899x+70.6840985836	0.089
M. euphorbiicola	CMM4867	y=15.3423755467x+79.6945427294	0.012
	CMM4868	y=13.2169312169x+82.5211640212	0.003
M. phaseolina (PCa)	CMM1531	y=14.5357142857x+77.8888888889	0.012
M. phaseolina (PCa)	MC01	y=16.6386225656x+76.0844032340	0.027
	Mean	-	0.034
	χ2 d	-	-

• ^a Positive control (PC).
• ^b y = percentage of mycelial growth inhibition for log-Probit analysis; and x = fungicide concentration.
• ^c Calculated by the concentration equation (mg L^-1) for logProbit analysis.

The carbendazim fungicide test of the three Macrophomina species showed that the EC₅₀ for all the isolates ranged from 0.003 to 0.089 mg L^-1. These isolates were considered sensitive to carbendazim (EC₅₀ < 0.1 mg L^-1). Edgington, Khew and Barrow (1971) proposed the following criteria to rank the fungitoxicity of a fungicidal substance: EC₅₀ < 1 mg L^-1 = highly fungitoxic, EC₅₀ of 1–50 mg L^-1 = moderately fungitoxic and EC₅₀ > 50 mg L^-1 = non-toxic. Thus, in this study, carbendazim was considered a highly fungitoxic chemical for all isolates of the studied species. Only a few studies have reported the sensitivity of M. phaseolina to carbendazim, but there are no studies in the literature that show the relationship of M. pseudophaseolina and M. euphorbiicola with carbendazim. This agrees with other studies that reported some degree of sensibility among M. phaseolina isolates to carbendazim (CHAUHAN, 1988). The carbendazim EC₅₀ values obtained in the present study were consistent with that of a previous study involving a Brazilian isolate of M. phaseolina from G. max (TONIN et al., 2013). Carbendazim belongs to the benzimidazole systemic fungicides group and is a potent inhibitor of tubulin polymerization and exerts its antifungal activity by targeting the β-tubulin subunit of the microtubules, which results in the arrest of microtubule formation and a failure in cell division, subsequently leading to cell death (FRAC, 2021). In relation to its chemical control, in Brazil, there are no fungicides registered for this pathogen in melon and watermelon crops (AGROFIT, 2022). In the present study, the EC₅₀ of the active ingredient carbendazim showed it to be a viable alternative for controlling Macrophomina spp. However, field studies will be necessary to evaluate the efficiency of this fungicide.

Relationships between adaptability components and pathogenicity of Macrophomina species

Cluster analysis using the UPGMA method was used to group Macrophomina species as a function of the Euclidean distance obtained from their adaptability components and pathogenicity (Figure 1). Three groups of Macrophomina species were formed. The first two groups were unitary, the first consisting of CMM4868 (M. euphorbiicola) and the second CMM1531 (M. phaseolina). The isolate CMM4868 was less sensitive to the fungicide since it had greater growth in almost all concentrations applied. However, it was more sensitive to salinity and less pathogenic to melon and watermelon. The CMM1531 isolate was more sensitive to the fungicide but its growth was little affected by salinity and temperature, mainly up to 30 ºC. It was pathogenic to melon.

Figure 1
Heatmap showing the grouping of Macrophomina isolates when analyzing the temperature, salinity, pathogenicity and fungicide (PWME, watermelon pathogenicity; S750, 750 mM salinity; S1000, 1000 mM salinity; F0.1, fungicide at the concentration 0.1 mg L^-1 a.i.; PMEL, melon pathogenicity; S250, 250 mM salinity; S500, 500 mM salinity; T25, the temperature at 25 ºC; T30, the temperature at 30 ºC; S0, salinity at 0 mM; T35, the temperature at 35 ºC; T40, the temperature at 40 ºC; F.1, fungicide at a concentration of 1 mg L^-1 a.i.; F.10, fungicide at a concentration of 10 mg L^-1 a.i.; F0.01, fungicide at a concentration of 0.01 mg L^-1 a.i.; F.100, fungicide at concentration 100 mg L^-1 a.i.). The dendrogram and order were determined using the functions of calculating the distance matrix (dist) and hierarchical cluster (hclust) in R. The red colours indicate higher rates of mycelial growth (temperature and salinity), a higher percentage of growth inhibition (fungicide) and higher disease severity (pathogenicity), while the white colours indicate lower growth rates of mycelial growth (temperature and salinity), a lower percentage of growth inhibition (fungicide) and lower disease severity (pathogenicity).

The third group brought together the other isolates that, in general, have their growth less influenced by the adaptability components (Figure 1). However, there was variation within the group. The isolate CMM4867 (M. euphorbiicola) showed similar behavior to the isolate CMM1531, that is, greater sensitivity to the fungicide and with greater growth in salinity up to 500 mg L^-1 (S500) and temperatures up to 30 ºC (T30). The isolate CMM4771 (M. pseuphaseolina) was less sensitive to the fungicide but affected by temperature and salinity. The isolate MC01 (M. phaseolina), as expected, was the most pathogenic to watermelon.

From the principal component analysis (PCA), it can be noted that the first component explains 33.40% of the data variability, while the second explains 24.06 %, totalling 57.46 % (Figure 2). The effects of the fungicide, especially the concentrations 1 mg L^-1 and 10 mg L^-1 , plus the higher temperatures, are the variables most associated with the first major component (CP1). On the other hand, salinity, especially concentrations 250 (S250) and 500 (S500), plus lower temperatures (25 and 30 ºC), are the variables most associated with the second main component (Figure 2).

Figure 2
Principal component analysis (PCA) of Macrophomina isolates with their adaptability components and pathogenicity (PWME, watermelon pathogenicity; S750, 750 mM salinity; S1000, 1000 mM salinity; F0.1, fungicide at the concentration 0.1 mg L^{- 1} a.i.; PMEL, melon pathogenicity; S250, salinity at 250 mM; S500, salinity at 500 mM; T25, temperature at 25 ºC; T30, temperature at 30 ºC; S0, salinity at 0 mM; T35, temperature at 35 ºC; T40, temperature at 40 ºC; F.1, fungicide at the concentration 1 mg L^-1 a.i.; F.10, fungicide at the concentration 10 mg L^-1 a.i.; F0.01, fungicide at the concentration 0.01 mg L^-1 a.i.; F.100, fungicide at a concentration of 100 mg L^-1 a.i.).

Regarding the distribution of species in relation to the adaptability components and pathogenicity, a result similar to that observed for the cluster analysis was observed. The CMM4868 isolate had greater growth when associated with different concentration of fungicide and higher temperatures but growth was reduced by lower salinity and temperatures. The CMM1531 isolate had greater growth in salinity concentration and was more affected by the fungicide. The isolate CMM4867 showed greater sensitivity to the fungicide but with greater growth in salinity and temperatures up to 30 ºC (T30). The isolate CMM4741 (M. pseudophaseolina) was less sensitive to the fungicide, but affected by temperature and salinity. The isolate MC01 stood out due to its greater pathogenicity to watermelon.

This work reports for the first time the association of M. euphorbiicola with asymptomatic roots of T. portulacastrum and B. diffusa weeds, which are common in the main Brazilian producing and exporting regions of melon and watermelon. The information generated in this research will increase knowledge about the epidemiology of disease and may help to predict the risk of Macrophomina root rot.

CONCLUSION

Macrophomina spp. used in these experiments showed growth variations “in vitro” for different temperatures, salinity and fungicide concentrations. All Macrophomina spp. used in these experiments were pathogenic to the tested melon and watermelon cultivars with the exception of the M. euphorbiicola isolates that did not cause damage to watermelon. Additional studies with new hybrids of these cultures should be carried out as a way to genetically manage these pathosystems in the studied region.

• Paper extracted from the thesis of the first author.

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Publication Dates

• Publication in this collection
  22 Aug 2022
• Date of issue
  Jul-Sep 2022

History

• Received
  02 Aug 2021
• Accepted
  08 Mar 2022